How selective antagonists and genetic modification have helped characterise the expression and functions of vascular P2Y receptors.

Vascular P2Y receptors mediate many effects, but the role of individual subtypes is often unclear. Here we discuss how subtype-selective antagonists and receptor knockout/knockdown have helped identify these roles in numerous species and vessels. P2Y1 receptor-mediated vasoconstriction and endothelium-dependent vasodilation have been characterised using the selective antagonists, MRS2179 and MRS2216, whilst AR-C118925XX, a P2Y2 receptor antagonist, reduced endothelium-dependent relaxation, and signalling evoked by UTP or fluid shear stress. P2Y2 receptor knockdown reduced endothelial signalling and endothelial P2Y2 receptor knockout produced hypertensive mice and abolished vasodilation elicited by an increase in flow. UTP-evoked vasoconstriction was also blocked by AR-C118925XX, but the effects of P2Y2 receptor knockout were complex. No P2Y4 receptor antagonists are available and P2Y4 knockout did not affect the vascular actions of UTP and UDP. The P2Y6 receptor antagonist, MRS2578, identified endothelial P2Y6 receptors mediating vasodilation, but receptor knockout had complex effects. MRS2578 also inhibited, and P2Y6 knockout abolished, contractions evoked by UDP. P2Y6 receptors contribute to the myogenic tone induced by a stepped increase in vascular perfusion pressure and possibly to the development of atherosclerosis. The P2Y11 receptor antagonists, NF157 and NF340, inhibited ATP-evoked signalling in human endothelial cells. Vasoconstriction mediated by P2Y12/P2Y13 and P2Y14 receptors was characterised using the antagonists, cangrelor, ticagrelor, AR-C67085 and MRS2211 or PPTN respectively. This has yet to be backed up by receptor knockout experiments. Thus, subtype-selective antagonists and receptor knockout/knockdown have helped identify which P2Y subtypes are functionally expressed in vascular smooth muscle and endothelial cells and the effects that they mediate.

Characterisation of P2Y receptor subtypes mediating vasodilation and vasoconstriction of rat pulmonary artery using selective antagonists.

Pulmonary vascular tone is modulated by nucleotides, but which P2 receptors mediate these actions is largely unclear. The aim of this study, therefore, was to use subtype-selective antagonists to determine the roles of individual P2Y receptor subtypes in nucleotide-evoked pulmonary vasodilation and vasoconstriction. Isometric tension was recorded from rat intrapulmonary artery rings (i.d. 200-500 µm) mounted on a wire myograph. Nucleotides evoked concentration- and endothelium-dependent vasodilation of precontracted tissues, but the concentration-response curves were shallow and did not reach a plateau. The selective P2Y2 antagonist, AR-C118925XX, inhibited uridine 5'-triphosphate (UTP)- but not adenosine 5'-triphosphate (ATP)-evoked relaxation, whereas the P2Y6 receptor antagonist, MRS2578, had no effect on UTP but inhibited relaxation elicited by uridine 5'-diphosphate (UDP). ATP-evoked relaxations were unaffected by the P2Y1 receptor antagonist, MRS2179, which substantially inhibited responses to adenosine 5'-diphosphate (ADP), and by the P2Y12/13 receptor antagonist, cangrelor, which potentiated responses to ADP. Both agonists were unaffected by CGS1593, an adenosine receptor antagonist. Finally, AR-C118925XX had no effect on vasoconstriction elicited by UTP or ATP at resting tone, although P2Y2 receptor mRNA was extracted from endothelium-denuded tissues using reverse transcription polymerase chain reaction with specific oligonucleotide primers. In conclusion, UTP elicits pulmonary vasodilation via P2Y2 receptors, whereas UDP acts at P2Y6 and ADP at P2Y1 receptors, respectively. How ATP induces vasodilation is unclear, but it does not involve P2Y1, P2Y2, P2Y12, P2Y13, or adenosine receptors. UTP- and ATP-evoked vasoconstriction was not mediated by P2Y2 receptors. Thus, this study advances our understanding of how nucleotides modulate pulmonary vascular tone.

Quality of life in an adolescent orthodontic population.

OBJECTIVES: To investigate adolescent orthodontic patient experiences and quality of life with fixed appliances compared to Invisalign. MATERIALS AND METHODS: Adolescent patients in active treatment with Invisalign or fixed appliances for a minimum of 6 months were provided with the Child Oral Health Impact Profile-Short Form 19 questionnaire, along with additional items of interest that were assessed separately. Pearson's χ2 test was used to compare responses (P < .05), and unpaired t-tests (P < .05) were used to test for differences in mean satisfaction, quality of life, and domain scores. RESULTS: In total, 74 patients (37 in each treatment group) participated. Overall, no significant differences were noted in the mean quality of life, satisfaction, or domain scores between the two groups. A significant difference was noted in the time taken to adjust to appliances, with the Invisalign group demonstrating faster adaptation. Additionally, the fixed appliance group was 3.8 times more likely to report missing school because of their appliance (95% confidence interval [CI]: 1.2, 12.5) and 2.7 times more likely to report having difficulty eating certain foods (95% CI: 1.1, 7.1). When the sample of females between the ages of 14 and 18 was analyzed, the Invisalign group reported feeling attractive more often than the fixed appliance group. CONCLUSIONS: Both treatment groups were generally very satisfied with their treatment modality. The overall quality of life of adolescent orthodontic patients undergoing treatment with fixed appliances and Invisalign for a minimum of 6 months was similar.

Investigation and comparison of patient experiences with removable functional appliances.

OBJECTIVES: To compare patients' experiences with the Invisalign Teen with Mandibular Advancement® (ITMA) and Twin Block (TB) appliances, both initially and after several months of wear. MATERIALS AND METHODS: Sixty-eight patients completed an anonymous survey after at least 2 months of wearing ITMA or TB. Forty-five patients treated with ITMA (18 boys, 27 girls, mean age 13.6 years, SD ± 1.54) and 23 patients treated with TB (13 boys, 10 girls, mean age 10.60 years, SD ± 1.92) were included. RESULTS: More patients using the TB found their appliance to be visually intimidating as compared with patients using the ITMA (21.7% vs 8.9%). TB was more noticeable than the ITMA (69.6% vs 25%). Appliance insertion was more difficult for TB patients (21.8% vs 4.44% for ITMA). After several months, there were more reports of tooth soreness and lip/cheek soreness in the ITMA group. TB patients were more embarrassed even after several months (14.3% vs 0% for ITMA). More TB patients required extra appointments for breakage (50% vs 22.2% for ITMA). Speech, drooling, and jaw and lip/cheek soreness worsened initially for both groups but improved over time. There were no differences between the groups regarding visible facial changes, satisfaction with treatment experience, or time to acclimatize to the appliance. CONCLUSIONS: TB and ITMA patients shared similar experiences for most of the parameters measured, but there were significant differences between the groups regarding appliance wear and management, discomfort, and function.

Comparison of early and conventional autogenous secondary alveolar bone graft in children with cleft lip and palate: A systematic review.

PURPOSE: The literature is scarce on studies comparing secondary alveolar bone graft (SABG) performed early at approximately 5-6 years and at the conventional time at 9-11 years. This systematic literature review(SLR) aimed to compare clinical outcomes after two different timings of SABG in children with unilateral and bilateral cleft lip and palate. METHODS: The inclusion criteria were autogenous iliac grafts and the following study designs: case control, cohort, clinical controlled trial (CCT), randomized CCT (RCCT), and previous SLRs. Ovid MEDLINE, Ovid EMBASE, Web of Science, Scopus, Cochrane, ProQuest and Google Scholar were the primary databases. Two calibrated examiners worked independently to select the articles. The MINORS evaluation method for surgical non-RCTs was used to assess for quality. RESULTS: 1,111 articles were retrieved and 19 qualified. Different clinical and radiographic outcomes such as bone level, periodontal status, canine eruption and cleft-side tooth survival were evaluated by different assessment methods such as CBCT volume, computed tomography, periodontal evaluation, panoramic, intraoral radiographs, and Bergland scale. No RCCT or meta-analysis was found. None of the studies received the ideal score, which is 16 for non-comparison studies and 24 for comparison studies. CONCLUSION: Methodological variation, lack of standardization for initial cleft dimension and low-quality level rendered a fair comparison unfeasible. Although further studies are necessary, it can be assumed that early SABG also can be an acceptable option, but this was based on a single study with a reasonable level of evidence.

Effect of Low Intensity Pulsed Ultrasound (LIPUS) on Tooth Movement and Root Resorption: A Prospective Multi-Center Randomized Controlled Trial.

The aim of this study was to evaluate the possible effect of low intensity pulsed ultrasound (LIPUS) on tooth movement and root resorption in orthodontic patients. Twenty-one patients were included in a split-mouth study design (group 1). Ten additional patients were included with no LIPUS device being used and this group was used as the negative control group (group 2). Group 1 patients were given LIPUS devices that were randomly assigned to right or left side on upper or lower arches. LIPUS was applied to the assigned side that was obtained by randomization, using transducers that produce ultrasound with a pulse frequency of 1.5 MHz, a pulse repetition rate of 1 kHz, and average output intensity of 30 mW/cm2. Cone-beam computed tomography (CBCT) images were taken before and after treatment. The extraction space dimensions were measured every four weeks and root lengths of canines were measured before and after treatment. The data were analyzed using paired t-test. The study outcome showed that the mean rate of tooth movement in LIPUS side was 0.266 ± 0.092 mm/week and on the control side was 0.232 ± 0.085 mm/week and the difference was statistically significant. LIPUS increased the rate of tooth movement by an average of 29%. For orthodontic root resorption, the LIPUS side (0.0092 ± 0.022 mm/week) showed a statistically significant decrease as compared to control side (0.0223 ± 0.022 mm/week). The LIPUS application accelerated tooth movement and minimized orthodontically induced tooth root resorption at the same time.

Characterisation of P2Y2 receptors in human vascular endothelial cells using AR-C118925XX, a competitive and selective P2Y2 antagonist.

BACKGROUND AND PURPOSE: There is a lack of potent, selective antagonists at most subtypes of P2Y receptor. The aims of this study were to characterise the pharmacological properties of the proposed P2Y2 receptor antagonist, AR-C118925XX, and then to use it to determine the role of P2Y2 receptors in the action of the P2Y2 agonist, UTP, in human vascular endothelial cells. EXPERIMENTAL APPROACH: Cell lines expressing native or recombinant P2Y receptors were superfused constantly, and agonist-induced changes in intracellular Ca2+ levels monitored using the Ca2+ -sensitive fluorescent indicator, Cal-520. This set-up enabled full agonist concentration-response curves to be constructed on a single population of cells. KEY RESULTS: UTP evoked a concentration-dependent rise in intracellular Ca2+ in 1321N1-hP2Y2 cells. AR-C118925XX (10 nM to 1 µM) had no effect per se on intracellular Ca2+ but shifted the UTP concentration-response curve progressively rightwards, with no change in maximum. The inhibition was fully reversible on washout. AR-C118925XX (1 µM) had no effect at native or recombinant hP2Y1 , hP2Y4 , rP2Y6 , or hP2Y11 receptors. Finally, in EAhy926 immortalised human vascular endothelial cells, AR-C118925XX (30 nM) shifted the UTP concentration-response curve rightwards, with no decrease in maximum. CONCLUSIONS AND IMPLICATIONS: AR-C118925XX is a potent, selective and reversible, competitive P2Y2 receptor antagonist, which inhibited responses mediated by endogenous P2Y2 receptors in human vascular endothelial cells. As the only P2Y2 -selective antagonist currently available, it will greatly enhance our ability to identify the functions of native P2Y2 receptors and their contribution to disease and dysfunction.

Hibiscus acid from Hibiscus sabdariffa (Malvaceae) has a vasorelaxant effect on the rat aorta.

Hibiscus sabdariffa (Malvaceae) is a plant that is widely recognised for its antihypertensive properties; however the constituent(s) responsible for this biological activity are presently unknown. The aim of this study was to identify the potential compounds that are responsible for the vasorelaxant activity of H. sabdariffa. Thereafter, the mechanisms involved in producing the vasorelaxation were investigated. The plant was extracted consecutively with hexane, ethyl acetate and methanol. The methanolic extract was subjected to bioassay-guided fractionation in order to isolate pure compounds that possessed vasorelaxant activity. The vascular effects of the pure compounds were studied on the rat aorta in vitro using myography techniques. Hibiscus acid produced a concentration-dependent relaxation of the rat aorta pre-contracted with either phenylephrine (3 µM) or KCl (60 mM), irrespective of the presence of the endothelium. When the tissue was pre-contracted with phenylephrine, the concentration required to produce 50% relaxation (IC50), was 0.09 ±â€¯0.01 mg/ml. Hibiscus acid had no effect on the phasic contraction induced by phenylephrine in Ca2+-free physiological solution; but it did affect the component of the contraction that is due to Ca2+ influx. In parallel studies, garcinia acid, a diastereoisomer of hibiscus acid, was found to have an almost identical vasorelaxant effect. The vasorelaxant action of both compounds is most likely due to the inhibition of Ca2+ influx via voltage-dependent Ca2+ channels.

Polarisation and functional characterisation of hepatocytes derived from human embryonic and mesenchymal stem cells.

Adult hepatocytes are polarised with their apical and basolateral membranes separated from neighbouring cells by tight junction proteins. Although efficient differentiation of pluripotent stem cells to hepatocytes has been achieved, the formation of proper polarisation in these cells has not been thoroughly investigated. In the present study, human embryonic stem cells (hESCs) and human mesenchymal stem cells (hMSCs) were differentiated to hepatocyte-like cells and the derived hepatocytes were characterised for mature hepatocyte markers. The secretion of hepatic proteins, expression of hepatic genes and the functional hepatic polarisation of stem cell-derived hepatocytes, foetal hepatocytes and the HepG2 hepatic cell line were evaluated and the different lines were compared. The results indicate that hESC-derived hepatocytes are phenotypically more robust and functionally more efficient compared with the hMSC-derived hepatocytes, suggesting their suitability for toxicity studies.

The Inhibitory Effect of Haloxylon salicornicum on Contraction of the Mouse Uterus.

Haloxylon salicornicum (H. salicornicum) is a plant that is frequently taken as a tea by Bedouin women in Egypt who are experiencing difficulties during pregnancy, as well as to provide relief from dysmenorrhoea. Despite its medical use, there has been no detailed evaluation of the effect of this plant on uterine tissue. Therefore, the initial aim of this study was to determine whether H. salicornicum affected the contraction of the mouse uterus in vitro. The crude aqueous extract of H. salicornicum was found to inhibit the spontaneous contractions of the uterus, with the effect being rapid in onset and completely reversible upon washout. Subsequent purification of the plant extract resulted in the identification of synephrine and N-methyltyramine, both of which were found to have inhibitory effects on the spontaneous contractions of the uterus. The EC50 for the purified constituent identified as synephrine was 0.82 ± 0.24 µ g/mL. The inhibitory activity of crude H. salicornicum, as well as the isolated constituents, could be prevented by pretreatment of the uterus with the ß -adrenoceptor antagonist propranolol. In conclusion, the use of H. salicornicum during preterm labour appears to be justified, and its pharmacologic effect is consistent with it acting as a ß -adrenoceptor agonist.

Using centrally held data to validate carotid surgery outcome data.

BACKGROUND AND PURPOSE: Outcome audit data for peer group comparison must be transparent, objective, and independently reproducible. Personal data sets are difficult to maintain and often lack complete follow-up. Local coding difficulties make initial retrieval of centrally held data unreliable. However starting with a complete list of interventions, reliable identification of patients who have experienced an adverse postoperative event may be possible using record linkages. METHODS: A surgical database, augmented by a hand-search of all theater registries and personal logbooks, identified 378 carotid endarterectomies performed for stroke prevention in symptomatic patients, in a single hospital between 2002 and 2009. A list of the names, unique patient identifiers, and operation dates was sent to the Information Services Division of National Health Service Scotland. Data were requested pertaining to all deaths and potential diagnoses of stroke after surgery. Every identified case was scrutinized. RESULTS: There were 30 (8%) readmissions or transfers of care identified within 30 days of surgery. From this, 12 strokes were identified with another 2 strokes, occurring without readmission, diagnosed in the outpatient clinic. Only 6 of the postoperative strokes were identified during the index admission. There were 2 early deaths resulting in a combined stroke and death rate of 4.2% (95% confidence intervals, 2.4%-6.9%). CONCLUSIONS: These outcome data are similar to the outcomes of the major carotid surgery trials. Record-linked data retrieval seems to be an appropriate starting point for outcome-based audit. This has the potential to generate robust, transparent data for comparison between individuals and centers for a specific procedure.

Suppression of epithelial-to-mesenchymal transitioning enhances ex vivo reprogramming of human exocrine pancreatic tissue toward functional insulin-producing ß-like cells.

Because of the lack of tissue available for islet transplantation, new sources of ß-cells have been sought for the treatment of type 1 diabetes. The aim of this study was to determine whether the human exocrine-enriched fraction from the islet isolation procedure could be reprogrammed to provide additional islet tissue for transplantation. The exocrine-enriched cells rapidly dedifferentiated in culture and grew as a mesenchymal monolayer. Genetic lineage tracing confirmed that these mesenchymal cells arose, in part, through a process of epithelial-to-mesenchymal transitioning (EMT). A protocol was developed whereby transduction of these mesenchymal cells with adenoviruses containing Pdx1, Ngn3, MafA, and Pax4 generated a population of cells that were enriched in glucagon-secreting α-like cells. Transdifferentiation or reprogramming toward insulin-secreting ß-cells was enhanced, however, when using unpassaged cells in combination with inhibition of EMT by inclusion of Rho-associated kinase (ROCK) and transforming growth factor-ß1 inhibitors. Resultant cells were able to secrete insulin in response to glucose and on transplantation were able to normalize blood glucose levels in streptozotocin diabetic NOD/SCID mice. In conclusion, reprogramming of human exocrine-enriched tissue can be best achieved using fresh material under conditions whereby EMT is inhibited, rather than allowing the culture to expand as a mesenchymal monolayer.